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Reto Crameri

Publications and source records attributed to Reto Crameri.

46 records · Page 3Linked to original sources
PubMed2002

Unfolding and double-stranded DNA binding of the cold shock protein homologue Cla h 8 from Cladosporium herbarum.

The cloning, purification, and biophysical characterization of the first eukaryotic cold shock protein homologue, Cla h 8, expressed as single functional polypeptide is reported here. It was discovered as a minor allergen of the mold Cladosporium herbarum by phage display using a library selectively enriched for IgE-binding proteins. Based on the sequence homology of Cla h 8 with bacterial cold shock proteins (CSPs), a homology-based computer model of the allergen was computed indicating an all-beta structure of Cla h 8. This major structural feature was confirmed by CD spectroscopy. Despite the structural similarities with bacterial CSPs, the DNA-binding and unfolding behavior of Cla h 8 exhibited unique and previously undescribed characteristics. High affinities of Cla h 8 for single-stranded DNA as well as for double-stranded DNA corresponding to the human Y-box were detected. The affinity for double-stranded DNA increased significantly with decreasing temperature, which was paralleled by an increase in the beta sheet content of the protein. Temperature-dependent fluorescence anisotropy and far-UV CD measurements revealed different unfolding transitions at 28 and at 35.7 degrees C, respectively, indicating a multistate transition, which is uncommon for CSPs. The enhanced affinity for DNA at low temperatures together with the low unfolding transition refer to the functional significance of Cla h 8 at reduced temperatures.

Allergens↗
PubMed2002

Comparison of the crystal structures of the human manganese superoxide dismutase and the homologous Aspergillus fumigatus allergen at 2-A resolution.

Manganese superoxide dismutase (MnSOD) of Aspergillus fumigatus, a fungus involved in many pulmonary complications, has been identified as IgE-binding protein. It has been shown also that MnSODs from other organisms, including human, are recognized by IgE Abs from individuals sensitized to A. fumigatus MnSOD. Comparison of the fungal and the human crystal structure should allow the identification of structural similarities responsible for IgE-mediated cross-reactivity. The three-dimensional structure of A. fumigatus MnSOD has been determined at 2-A resolution by x-ray diffraction analysis. Crystals belonged to space group P2(1)2(1)2(1) with unit cell dimensions of a = 65.88 A, b = 98.7 A, and c = 139.28 A. The structure was solved by molecular replacement using the structure of the human MnSOD as a search model. The final refined model included four chains of 199-200 amino acids, four manganese ions, and 745 water molecules, with a crystallographic R-factor of 19.4% and a free R-factor of 23.3%. Like MnSODs of other eukaryotic organisms, A. fumigatus MnSOD forms a homotetramer with the manganese ions coordinated by three histidines, one aspartic acid, and one water molecule. The fungal and the human MnSOD share high similarity on the level of both primary and tertiary structure. We identified conserved amino acids that are solvent exposed in the fungal and the human crystal structure and are therefore potentially involved in IgE-mediated cross-reactivity.

Allergens↗
PubMed2002

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment.

Type I allergy is an immunoglobulin E (IgE)-mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen-containing sources but cannot identify the disease-eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients' IgE reactivity profiles to large numbers of disease-causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.

Allergens↗
PubMed2002

Cyclophilins, a new family of cross-reactive allergens.

Type I allergic reactions occur by immediate release of anaphylactogenic mediators due to cross-linking of IgE bound to the high-affinity Fc(epsilon)RI on the surface of effector cells of sensitized individuals after allergen exposure. IgE-mediated hypersensitivity against normally innocuous environmental antigens is of clinical importance because of an increasing incidence of asthma and severe atopic diseases causing raising health care burdens to the society. A vast variety of different molecular structures has been shown to be able to induce hypersensitivity reactions. However, the high structural homology between phylogenetically conserved allergenic proteins present in different, apparently unrelated sources of exposure seems to play an important role in IgE-mediated poly-sensitization. These allergen families, formally termed pan-allergens, represent proteins sharing a high degree of sequence homology. Here we report cloning, production and serological investigations of a new pan-allergen family, the cyclophilins, found to be cross-reactive across species including humans. IgE-mediated cross-reactivity against autoantigens may contribute to perpetuation of severe atopic disorders even in the absence of exogenous allergen exposure. The molecular definition of pan-allergen families may substantially contribute to reduce the number of structures needed for diagnosis and therapy of allergic diseases based on highly pure, standardized recombinant allergens.

Allergens↗
PubMed2002

T cells and eosinophils cooperate in the induction of bronchial epithelial cell apoptosis in asthma.

BACKGROUND: Asthma is an inflammatory airway disease associated with an infiltration of T cells and eosinophils, increased levels of pro-inflammatory cytokines, and shedding of bronchial epithelial cells (ECs). OBJECTIVE: Shedding of bronchial ECs is characterized by loss of the normal bronchial pseudostratified epithelium and the maintenance of a few basal cells on a thickened basement membrane. The aim of this study was to investigate whether, and by which mechanism, T cells and eosinophils can cause damage to airway ECs. METHODS: Bronchial ECs, cultured and exposed to cytokines, eosinophil cationic protein, activated T cells, and eosinophils were studied for the expression of apoptosis receptors (flow cytometry, immunoblotting, and RNA expression) and for the susceptibility for undergoing apoptosis. In addition, bronchial biopsy specimens from patients with asthma were evaluated for EC apoptosis. RESULTS: We demonstrate herein that the respiratory epithelium is an essential target of the inflammatory attack by T cells and eosinophils. Bronchial ECs underwent cytokine-induced cell death with DNA fragmentation and morphologic characteristics of apoptosis mediated by activated T cells and eosinophils. T cell- and eosinophil-induced EC apoptosis was blocked by inhibition of IFN-alpha and TNF-alpha; the Fas ligand-Fas pathway appears to be less important. Recombinant eosinophil cationic protein induced mainly necrosis of ECs. Furthermore, we demonstrated in situ apoptotic features of ECs in bronchial biopsy specimens of asthmatic patients. CONCLUSION: T cell- and eosinophil-induced apoptosis represents a key pathogenic event leading to EC shedding in asthma.

Apoptosis↗
PubMed2002

Phage display based cloning of proteins interacting with the cytoplasmic tail of membrane immunoglobulins.

The reduced quantity and quality of serum immunoglobulins (sIgs) in mutant mice expressing truncated cytoplasmic tails of IgE and IgG1 indicate an active role for the cytoplasmic domains of mIgG1 and mIgE. We used phage display technology to identify candidate proteins able to interact with the cytoplasmic tail of mIgE. Using a murine cDNA B cell library displayed on the surface of phage as prey and the 28 amino acid long cytoplasmic tail of IgE as bait, we isolated phage encoding the murine hematopoietic progenitor kinase 1 (HPK1). Surface plasmon resonance analysis measurements confirmed affinity of HPK1 to the mIgE cytoplasmic tail and revealed association to other immunoglobulin isotypes as well. Immunoprecipitation experiments, using lysates from two B cell lines expressing nitrophenyl (NP) specific mIgE molecules showed co-precipitation of IgE and HPK1. The interaction of HPK1 with the cytoplasmic domains of membrane immunoglobulins indicate an active role of the tails as part of an isotype specific signal transduction, independent from the Igalpha/Igbeta heterodimers, and may represent a missing link to upstream regulatory elements of HPK1 activation.

Amino Acid Sequence↗
PubMed2002

Immunological and structural analysis of IgE-mediated cross-reactivity between manganese superoxide dismutases.

BACKGROUND: Allergy results from inappropriate immune responses to normally innocuous proteins. More than 300 IgE-binding proteins have been cloned and shown to cover structurally and functionally heterogeneous protein families including enzymes, backbone and storage proteins as well as proteins with unknown function. Structurally related cross-reactive allergens are involved in the pathogenesis of important clinical syndromes. We found that phylogenetically highly conserved proteins, including human manganese superoxide dismutase (MnSOD), are involved in IgE-mediated autoreactivity. METHODS: MnSOD cloned from different phylogenetically distant species were produced as recombinant proteins in Escherichia coli and used to study IgE-mediated cross-reactivity and proliferative responses in peripheral blood mononuclear cells (PBMC) of individuals sensitised to Aspergillus fumigatus MnSOD. Homology models of the three-dimensional structures of MnSOD from A. fumigatus, Drosophila melanogaster and Saccharomyces cerevisiae using the human MnSOD structure refined at 2.2 A as template were constructed to identify conserved amino acid residues exposed to the solvent. RESULTS: Cross-reactivity between the MnSOD at B and T cell level was demonstrated by inhibition experiments showing shared B cell epitopes and by the capability of the different MnSODs to induce proliferative responses in PBMCs of sensitised individuals. Structural modelling allowed to identify conserved residues exposed to the solvent. The identified residues are scattered over the sequence of the enzyme indicating putative conformational IgE-binding epitopes. CONCLUSIONS: The results obtained corroborate molecular mimicry as a plausible mechanism to explain autoreactivity to human MnSOD. Moreover, homology modelling provides a rational tool to identify conserved residues involved in defining cross-reactive B cell epitopes.

Adult↗
PubMed2002

Direct selection of cDNAs from filamentous phage surface display libraries: potential and limitations.

Over the past decade, powerful technologies devoted to survey large molecular libraries for the presence of specific clones using the discriminative power of affinity selection have been developed. Phage surface display technology is the most established of these methods and has revolutionised our ability to select agonistic and antagonistic peptides, antibodies with desired specificity and other drug targets. Thereby ligands are expressed as fusions to phage coat proteins and their respective genes are packaged within the phage. The basic concept of linking the phenotype, expressed as gene product displayed on the phage coat, to its genetic information integrated into the phage genome, creates fusion proteins covalently associated with the infectious particle itself. Binding of the phage to the target molecule offers a selective system by which rare phage carrying the desired gene product can be selected from large phage populations carrying inappropriate sequences. Phage selected in this fashion can be used for subsequent rounds of selection because they are able to self-replicate in suitable host cells, yielding target-specific phage populations after several consecutive rounds of affinity selection. Over 1500 publications describe the use of phage display technology highlighting its performance. Phage display possesses certain limitations, including its use for selection of genes from cDNA libraries that has lagged behind, despite the many accomplishments of this technology. Here we discuss recent progress in construction and screening of cDNA libraries displayed on phage surface and emerging concepts allowing fast identification of virtually all different clones present in enriched libraries.

Bacteriophages↗
PubMed1998

[Development of a pyrogen test based on rabbit blood]

The testing of drugs for pyrogen contamination e.g. the exclusion of fever inducing impurities, was standardised in the Forties in the form of animal pyrogen assay. We recently proposed an alternative method using human whole blood from healthy donors. The substances to be tested are incubated in the presence of blood and the generation of endogenous pyrogens (Interleukin 1, IL-1) are measured reflecting the primary fever reaction in man. With this method it is possible to detect the reaction of the relevant species. However, pronounced differences were reported for the potency of several bacterial pyrogens in different species (e.g. up to factor 10.000 for an individual endotoxin in different mammals). As a part of the validation of the new method discrepancies between the whole blood system and the rabbit test might occur. In these cases it is not possible to distinguish between an in-vitro-artefact and species differences. Therefore, the goal of this project is the development of an in-vitro-test based on rabbit blood. For this purpose rabbit IL-1 was expressed. At present chickens and mice are immunized and possibly crossreacting anti-human-IL-1-antibodies are tested. In this way, an ELISA for the rabbit cytokin will be established. Using a temporarily commercially available rabbit TNF-ELISA, the feasibility of the test-system could already be shown: Pyrogens induced the generation of TNF in rabbit blood. In the human model, the endpoints IL-1 and TNF were very similar. The sensitivity of human blood and rabbit blood appeared to be very similar. However, individual pyrogens (only different endotoxins were tested up to now) showed considerable deviations. However, these results require the verification in the final test. The rabbit test in preparation will be implemented immediately into the ongoing evaluations by the Paul-Ehrlich-Institute (BMBF) and at the University of Heidelberg (European Pharmakopoea). Furthermore, such a test also represents an interesting alternative in the veterinarian medical field using the correct target species for pyrogenicity testing. This project war supported by the Foundation Research 3R, CH-Muensingen.

Journal Article↗